We want to learn how Gram-positive bacteria control their synthesis of secondary metabolites -- substances that they produce only after vegetative growth and cell division have stopped. Such products include antibiotics, toxins, a number of enzymes, and a variety of small molecules. We will use as a model system the production of acetoin (2-oxo, 3-hydroxy-butane) by Bacillus subtilis. This compound is excreted by stationary-phase cells in large quantities. It comes from the decarboxylation of acetolactate, which the bacteria products by means of an enzyme, acetolactate synthase (AlsS), that is synthesized only after growth has stopped. We have cloned and sequenced the alsS gene that encodes AlsS, and an adjacent gene orfX that is transcribed and translated as a divergent operon. AlsA has sequence homology with IlvB, a biosynthetic enzyme of B. subtilis required for the synthesis of branched-chain amino acids. OrfX has sequence homology with a family of positive regulatory proteins found in other bacteria. We want to learn how the alsS gene's expression is controlled so that its product is not detectable during vegetative growth, but is produced once growth stops. We suspect that the orfX gene is involved in this control, but do not have direct evidence for this. A mutation, alsAl, unlinked to alsS, prevents the expression of alsS under all conditions. We have cloned alsA and will determine the role it plays in controlling the expression of alsS.